Two-color (two-photon) excitation (2CE) microscopy has been proposed [Lindek, S. and E. Stelzer, Opt Left 24 (1999), 1505-1507] where a specimen is excited by pair of photons of different wavelengths λ1 and λ2. The single-photon excitation (1P) wavelength λe of the sample is related to λ1 and λ2 according to: 1/λe=1/λ1+1/λ2. 2CE may be implemented with two confocal excitation beams that make an angle θ with respect to each other. Two-photon excitation (2PE) is a special case of 2CF microscopy where: λ1=λ2=2λe=λ2p.
The implementation of 2CE is seriously hindered by the lack of a suitable light source that permits for an efficient two-color excitation. 2CE with λ1=380 nm and λ2=780 nm, has been reported earlier [Lakowicz, J., et al., J. Phys. Chem., 100 (1996), 19406-19411] with a cavity-dumped dye laser which is an excitation source that is difficult to adapt in a 2CF microscope set-up.
We have discovered a new and efficient method of achieving 2CE with two confocal excitation beams via a Raman shifter as a single light source for both λ1 and λ2. 2CE is demonstrated in a Coumarin 6H (C15H15NO2) sample using the first two Stokes outputs (λ1=683 nm, λ2=954 nm) of the Raman shifter.
2CE with focused excitation beam(s) and a Raman shifter as light source has not yet been reported. A previous work by Uesugi et al. [J Raman Spectrosc. 31(4) (2000), 339-348] utilized two-color excitation (λ1=525 nm, λ2=560 nm) with a collimated beam from a Raman laser and only for excitation/absorption studies.